Aurelia Gaeta

Monitoring for cytomegalovirus infection in organ transplant recipients: Analysis of pp65 antigen, DNA and late mRNA in peripheral blood leukocytes

The use of sensitive and specific methods for rapid and reliable diagnosis is required due to the considerable impact of human cytomegalovirus (HCMV) in organ transplant recipients. For this purpose the demonstration of the presence of viral antigens in peripheral blood leukocytes (PMNLs) and of viral nucleic acids in the same cells or in sera would seem to be of valid support. The present study was designed to test pp65 antigen, HCMV DNA and HCMV late mRNA in order to provide clinical information for the management of the infection. Fifty solid organ recipients were monitored for six months after transplant. The data obtained from the various tests were analysed from the first evidence of HCMV infection revealed by positive antigenaemia and/or DNA‐polymerase chain reaction (PCR). In 3 asymptomatic and in 7 symptomatic patients, PCR became positive 1–2 weeks before antigenaemia but PCR did not discriminate the clinical evolution of HCMV infection. The antigenaemia test well correlated to the development of viral infection being positive in all symptomatics and in 31, 2% of asymptomatics. The antigenic load >100/2 × 105 positive cells was always associated with clinical signs of illness. The detection of late mRNA was more indicative of the virus replicative status in the follow‐up of patients treated with ganciclovir. In some cases there was evidence, prior to the other two tests, the block of viral replication due to the antiviral therapy and in others the onset of HCMV infection relapse. J. Med. Virol. 53:189–195, 1997.

Inhibition of in vitro proliferation of Epstein Barr virus infected B cells by an antisense oligodeoxynucleotide targeted against EBV latent membrane protein LMP1

Inhibition of in vitro proliferation of Epstein Barr Virus infected B cells by an antisense oligodeoxynucleotide targeted against EBV latent membrane protein LMP1

Multiplex polymerase chain reaction for the evaluation of cytomegalovirus DNA load in organ transplant recipients

Because of the considerable impact of human cytomegalovirus (HCMV) infection, sensitive, specific, and standardized methods are required for rapid and accurate evaluation of viral load in monitoring transplant recipients. The aim of the present study was to evaluate the usefulness of a multiplex polymerase chain reaction (PCR) for the coamplification of HCMV‐DNA and β‐globin genomic sequence in polymorphonuclear leukocytes (PMNL). Analysis and quantification of PCR products were carried out by a DNA enzyme immunoassay (DEIA), which is based on the hybridization of amplified DNA with a single‐stranded DNA probe, which coats microtitre wells. Colorimetric detection of the DNA‐antibody complex was carried out and optical density (O.D.) was recorded at 450/630 nm. To quantify HCMV/DNA load, a standard curve to which samples O.D. refer was obtained by amplifying serial dilutions of recombinant pGEM‐3Z plasmid DNA containing a genomic fragment of glycoprotein B. 340 PMNL specimens from 102 solid organ recipients were tested for the detection of pp65 antigen and HCMV‐DNA. The results showed a good correlation between viral load and clinical symptoms of HCMV infection; high specificity and predictive values for HCMV disease were found by PCR, using a cut‐off limit of 103 genomic copies per 2 × 105 PMNL. These findings indicate that the system described is an efficient and reproducible diagnostic method easy to apply for routine diagnosis and therapeutic monitoring of transplanted patients. J. Med. Virol. 61:251–258, 2000.

Early collection of saliva specimens from Bell's palsy patients: quantitative analysis of HHV-6, HSV-1, and VZV

Bells palsy is the most common cause of facial paralysis. Although it has been associated with diabetes mellitus, hypertension, pregnancy, and preeclampsia, the etiology of Bells palsy remains unknown. The reactivation of latent herpes simplex virus (HSV) or varicella‐zoster virus (VZV) with subsequent inflammation and entrapment of the facial nerve in the narrow labyrinthine segment has been implicated as a cause of facial paralysis, but the active role of these viruses in Bells palsy is still discussed. This study quantified HSV‐1 DNA, VZV DNA, and HHV‐6 DNA in 95 saliva samples collected from patients within 48 hr from the onset of paralysis. HSV‐1, VZV, and HHV‐6 were detected in 13%, 3%, and 61% of patients, respectively. The detection rate did not differ significantly between patients and a control group of healthy donors. Interestingly, however, the value of HHV‐6 DNA copies was significantly higher than that detected in healthy donors. In addition, the mean value of HHV‐6 DNA recorded in patients who had at least a one grade improvement of palsy at the first visit was significantly lower than that detected in patients who showed no change in facial palsy grade or an increase of at least one grade. These findings call into question the role of HSV‐1 and VZV in the etiology of Bells palsy, and suggest that HHV‐6 may be involved in the development of the disease or that the underlying disease mechanism might predispose patients to HHV‐6 reactivation. J. Med. Virol. 86: 1752–1758, 2014.

Ganciclovir penetrates into the cerebrospinal fluid of an infant with congenital cytomegalovirus infection.

Currently, there is no evidence whether ganciclovir, or its oral prodrug valganciclovir, penetrates into the cerebrospinal fluid of human infants treated for congenital cytomegalovirus infection. Here, we report a case study providing evidence that ganciclovir, administered as valganciclovir, reaches the infant’s cerebrospinal fluid when used at the currently recommended dose for congenital cytomegalovirus infection.

Opsonic activity of intravenous immune globulin on Gram-negative bacteria exposed to a monobactam antibiotic

Abstract Sub-inhibitory concentrations of antibiotics can alter the morphology and structure of the bacterial surface leading to better opsonization and therefore enhanced phagocytosis. The opsonic activity of a standard intravenous immune gamma globulin (IVIG) was tested for control bacteria and bacteria grown in the presence of sub-minimal inhibitory concentration (sub-MIC) of aztreonam, and β-lactam antibiotic. IVIG enhanced uptake by polymorphonuclear leucocytes of five Gram-negative bacteria. This enhancement was further increased when bacteria were exposed to sub-MIC of aztreonam.

Automated Intelligent Microscopy for the Recognition of Decoy Cells in Urine Samples of Kidney Transplant Patients

BACKGROUND BK virus (BKV)-associated nephropathy is definitely involved in allograft failure after kidney transplant. Thus, the need for an early control of viral reactivation in immunocompromised patients is well established. Determination of urinary release of decoy cells (DC) and BK viral load in plasma and urine by polymerase chain reaction (PCR) usually precedes renal biopsy. The aim of the study is to assess viral reactivation by BKV-DNA PCR and DC detection in urinary sediment using automated intelligent microscopy. METHODS Seventy-eight kidney transplant patients were analyzed for the presence of plasma BKV-DNA by quantitative TaqMan real-time PCR. Additionally, automated intelligent microscopy was used for urine sediment analysis, allowing to count cells with decoy feature, confirmed by phase contrast microscopic review. RESULTS Plasma BKV-DNA PCR was detected in 14 (17.9%) patients. DC were identified in 19 (24.3%) urine sediments by automated analyzers and confirmed by microscopic observation. Two patients were BKV-DNA-positive/DC-negative; conversely, 7 subjects were DC-positive/BKV-DNA-negative. CONCLUSIONS Plasma quantification of BK viral load is currently the best noninvasive method for the detection of viral reactivation. Nevertheless, automated methods to screen for the presence of DC in urine could facilitate early BK virus replication diagnosis and patient follow-up by quantitative and visual results.